Disulfide bond exchange in rhodopsin.
Rhodopsin contains two cysteines (Cys110 and Cys187) that are highly conserved among members of the G protein coupled receptor family and that form a disulfide bond connecting helixes 3 and 4 on the extracellular side of the protein. However, recent work on a rhodopsin mutant split in the cytoplasmic loop connecting helixes 3 and 4 has shown that the amino- and carboxy-terminal fragments of this split protein do not comigrate on nonreducing SDS-PAGE gels, suggesting that the native Cys110-Cys187 disulfide bond is not present in this mutant [Ridge et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3204-3208; Yu et al. (1995) Biochemistry 34, 14963-14969]. We show here that the inability to observe the disulfide bond on SDS gels is the result of a disulfide bond exchange reaction which occurs when this split rhodopsin is denatured in preparation for SDS-PAGE. Cys185 reacts with the native disulfide, displacing Cys110 and forming a new disulfide with Cys187. If the sulfhydryl-specific reagent N-ethylmaleimide is included in the sample during preparation for electrophoresis or if Cys185 is changed to Ser, the two fragments do comigrate with full-length rhodopsin on SDS gels and, therefore, are connected by the native Cys110-Cys187 disulfide bond. In related experiments, we find no evidence that the Cys110-Cys187 disulfide bond is broken upon formation of the active intermediate metarhodopsin II.
Kono
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M
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
Yu
H
H
Oprian
D D
DD
D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide; D016296:Mutagenesis; D010782:Photolysis; D011489:Protein Denaturation; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism
D004220:Disulfides; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine; D005033:Ethylmaleimide